Molecular Mechanism Of Nedd4 - Like E3 Ubiquitin Ligase In The Regulation Of Angiomotin / P130 Protein Stability

Angiomotin (AMOT) is a membrane-associated protein that is expressed in endothelial cells (ECs) and controls migration, tight junction (TJ) formation, cell polarity and angiogenesis. Recent studies also revealed AMOT, two AMOT-like proteins AMITL1 and AMOTL2 all play critical roles in Hippo pathway by regulating the subcellular localization of co-activators YAP/TAZ.However, it has been unclear how AMOT is regulated at cellular context. Here, we report that AMOT undergoes proteasomal degradation in cells. When 293T cells were treated with the proteasome inhibitor MG132 or lactacystin, AMOT protein rapidly increased. We searched for the relevant E3 ligase that was responsible for AMOT degradation. Sequence alignment of multiple AMOT orthologs across different species revealed that three L/P-P-X-Y motifs at the N-terminal region of AMOT/p130, AMOTL1 and AMOTL2 (except AMOT/p80) are highly conserved. L/P-P-X-Y motifs are known to interact with WW domain-containing proteins, which includes Nedd4-like E3 ubiquitin ligases family. So we screen a panel of several members of Nedd4 E3 ubiquitin ligases family. And we identify three members of Nedd4-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130, but not the short isoform of AMOT, AMOT/p80. Our data showed that Nedd4, Nedd4-2 and Itch could efficiently promote AMOT/p130 degradation in a dose-dependent manner, and could also efficiently promote AMOT L1 (but not AMOT L2) degradation.We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 and Itch lead to AMOT/p130 proteasomal degradation. We found HECT domain of Nedd4, Nedd4-2 and Itch and their ubiquitin ligase activity are required for promoting AMOT/p130 degradation. AMOT/p130 degradation induced by Nedd4 co-expression could be completely rescued by the proteasome inhibitor MG132. Wild type Nedd4 but not the Nedd4 CA mutant promotes AMOT/p130 protein turnover. Overexpression of Nedd4 leads to endogenous AMOT/p130 proteasomal degradation.While Knockdown of Nedd4, Nedd4-2 and Itch by small interfering RNA causes an accumulation of steady-state level of AMOT/pl30. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might positively regulate Hippo pathway through competing with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation.These observations provide a potential link between Nedd4-like ubiquitin ligases and Hippo pathway.